Extraction of steroid conjugates with a neutral resin

265

EXTRACTION OF STEROID CONJUGATES WITH A NEUTRAL RESIN

H. Leon Bradlow Institute for Steroid Research, Montefiore Hospital and Medical Center, New York, N. Y. 10467

Received December 4, 1967

ABSTRACT An improved and simplified procedure for the extraction of steroid conjugates from urine, s described. Following ads®rption of the conjugates on Arnberlite XAD-2k~J (a neutral cross linked polymer) from urine, the conjugates were readily eluted with methanol or ethanol. Recovery was substantially quantitative (greater than 90%) for the metabolites derived from cortisoi, testosterone, progesterone, estradiol and aldosterone. It was demonstrated that cortisol metabolites are not altered by adsorption on the resin. Many procedures (1-10) have been advanced for the extraction of

conjugated steroid hormone metabolites from urine and bile. One or more of the following objections limit existing methods: 1) large volumes of solvent, 2) low pH and the possibility of altering the steroids, 3) cumbersome manipulative procedures, and 4) poor recovery. Amberlite XAD-2*, a neutral cross linked polystyrene polymer was found to adsorb conjugates of steroid metabolites *

quantitatively from

Amberlite XAD-2 was obtained from the Rohm and Haas Co., Philadelphia, Pa. in the form of hydrated beads with an effective size of 0.3-0.45 ram.

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urine. The conjugates were readily eluted from the resin with methanol or ethanol. EXPERIMENTAL Typica I procedure: A column, 7 x 35 cm with a coarse flitted disc layered with 1 - 2 cm oF sand, was half filled with water. One kg of Amberlite XAD-2, slurried in water, was poured into the column. The resin was washed by flowing distilled water from the bottom of the column at a rate just sufficient to keep the resin suspended. The overflow was removed by a tube attached to a water aspirator. As soon as all the fines had been removed from the top (15-20 minutes) and the water had drained from the column it was ready for use. Two liters of urine were percolated through the resin under gravity flow (45 - 60 minutes), followed by 4 liters of water which was allowed to drain completely. The conjugates were eluted portionwise with 5 liters of methanol. The methanol was concentrated in vacuo in a flash evaporator. The combined residue usually contained 10% or less of the urine solids. The material was soluble in water, alcohol and tetrahydrofuran among other solvents. The column was regenerated by washing with water as described above, and used repeatedly with no significant loss of capacity. The column should be wet when not in use because the resin deteriorates on drying. With a single 1 kg column 4 liters of urine can be processed in a day with only occasional attention. Representative results are given in Table 1. The urine samples us~=d for these studies were obtained from patients who had received labeled steroid hormones in the course of other studies in this laboratory. Aliquots of all

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TABLE I RECOVERY OF RADIOACTIVITY FROM URINE FOLLOWING CHROMATOGRAPHY ON XAD-2 COLUMN

Typical

Examples %of

Fraction Study

Radioactivity

Cortisol Metabolites

TestosteroneMetabolites

1

2

1

0.2

0.4

0.4

0.2

0.4

1.1

0.7

0.4

1

78.8

84.3

94.2

79.8

2

15.9

15.2

3.5

16.0

3

3.5

3.0

1.3

5.5

4

1.6

0.7

0.5

1.5

5

0.9

0.4

0.4

1.0

101.1

103.4

Urine Effluent

2

Eluant Water

Methanol

101.3

104.1

One Kg of XAD-2 in a 7 x 35 cm column was used. Two liters of urine were percolated through the column, and then washed with 4 liters of water. The methanol eluant was collected in liter portions.

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fractions were counted to determine the recovery. Experiments were carried out comparing methanol, ethanol, t-butanol, tetrahydrofuran, and acetone as eluting solvent. Methanol, ethanol, and tetrahydrofuran were equally satisfactory. Acetone gave a poor recovery of radioactivity, and t-butanol flowed too m

slowly through the

column.

After ~-glucuronidase hydrolysis or solvolysis and extraction of the free steroids with ether, the unhydrolysed conjugates remaining in the urine could also be quantitatively extracted by this procedure. In seven urines treated in this manner the recovery in the methanolic extract was 90-99% following adsorption on XAD-2; only 0.1-2.7% was found in the aqueous effluents= Because of the low specific gravity of the resin (1.03) it was necessary to dilute some of these treated urines because adsorption is inefficient if the resin floats. The recovery of conjugates from these previously treated urines was not affected by pH. Equally good adsorption was obtained from strongly acid or alkaline solutions. The conjugates could not be eluted from the resin by washing with aqueous 0.]N acid or base. Small organic molecules like urea and glucose are not retained by the resin and are quantitatively recovered in the aqueous effluent. In order to determine whether artifact formation occurred during the extraction procedure, urine obtained from a patient who had been given cortisol-4-14C i.v. was divided into two portions. One portion was directly hydrolysed with 300 units/ml of Ketodase** for 5 days at 37° followed by continuous ether extraction for 48 hrs (11). The conjugates were extracted from the other aliquot with XAD-2. The methanol was concentrated in vacuo **

Warner Chilcott Leboratory, N. J.

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ST ER O I D S

and the residue was dissolved in the original volume of water and hydrelysed under the same conditions as the original urine. Portions of each neutral extract were chromatographed on 118 x 18 cm strips of Whatman #1 paper for 24 hrs in the system benzene:methanohethyl acetate: water (1:1-0.1:1). The strips were scanned on a Vanguard 880 scanner. The leper areas corresponding to the various peaks were eluted with alcohol and counted. The same metabolite spectrum was obtained by both procedures as shown in Table II. The capacity of the resin for steroid conjugates was studied using aqueous solutions of the sodium salts of synthetic dehydroisoandrosterone sulfate, cortisol phosphate, cortisol hemisuccinate and the conjugates in the urine of a patient who had received 160 mg//day of cortisol-4-14C. The recovery was nearly quantitative from the urine of the patient who had received the large dose of cortisol. When the synthetic conjugates were studied the ratio of conjugates to resin was found to be important. The recovery was quantitative for dehydroisoandrosterone sulfate and cortisol hemisuccinate when the ratio of conjugate to resin was less than 50 mg per 100 g of resin. At higher concentrations a substantial portion of the conjugate was recovered in the initial aqueous effluent. For cortisol phosphate the recovery was only satisfactory at ratios below 25 mg/100 g of resin. All samples were counted in a Packard 3365 liquid scintillation counter using the diotol scintillant mixture described by Herberg (12). RESULTS AND DISCUSSION Table ! summarizes typical results. Only a minor fraction of the total radioactivity remained in the urine after percolation through the XAD-2

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TABLE II RECOVERY OF METABOLITES OF CORTISOL-4-14C FOLLOWING XAD-2 TREATMENT

Metabolltes****

cortols

Control XAD-2* % of Extract 6.8

8.8

cortolones

16

15

THF

24

20

allo THF THE

7.5 24

7.1 20.

*The urine was divided in two portions. One was directly hydrolysed with 300 units/ml of Ketodase for 5 days at 37° followed by continuous ether extraction. The other was extracted by the XAD-2 procedure. The extract was concentrated, dissolved in water and hydrolysed under the same conditions. The yield of neutral extract was essentially identical, 67 and 70% respectively. cortols = pregnane-3ol, 11/3,17,20~and/3, 21 pentol cortolones = 11-ketopregnane-3c~,17,20~ and/3, 21 tetrol TH F = 20-ketopregna ne-3o~,11/3,17,21-tetrol allo THF = 20-keto-Sc~-pregnane-3c~,11/3,17,21-tetrol THE = 11,20-diketopregnane-3~, 17,21-triol

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STEROIDS

2"/1

column. The methanol rapidly eluted the steroid conjugates with the greatest part being recovered in the first volume of solvent. Good results were obtained for the recovery of different urinary conjugates derived from a variety of steroid tracers following their administration to man. Following progesterone 90-99% in 3 studies; cortisol 92-104% in 9 studies; testosterone 91-103% in 5 studies;

aldosterone 95%; and estradlol 90%. The experiments with synthetic conjugates show that recovery is satisfactory at levels up to 250 mg of steroid conjugates per liter of urine. In addition to the high recovery of coniugates the procedure is easier and more convenient than others reported in the literature. The procedure can also be applied to urines containing free steroids in addition to conjugated metabolites= Unconjugated or free cortisol was quantitatively adsorbed by the resin when carried through the standard procedure. Other investigators have recently reported the use of Amberlite XAD-2 for the extraction of urinary steroid conjugates with equally good results***. ***

Private communications from Drs. K. Kozuma, W. Kelly, and M. Levitz. While this work was in progress, Mattox and Vrieze (Fed. Proc., 26//2 Abstract #945, 1967) described the use of this resin in the isolation of synthetic steroid glucuronides.

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ACKNOWLEDGMENTS

The interest and support of Dr. T. F. Gallagher are gratefully acknowledged= Thanks are due Miss Ann Powell and Mrs. Gertrude Gilman for skillful assistance. This work was supported by a grant from the American Cancer Society and by Grants CA-07304 from the National Cancer Institute and FR-53 from the General Clinical Research Centers Branch, National Institutes of Health, U.S.P.H.S.

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Jayle, M. F. (ed), ANALYSE DES STEROIDES HORMONAUX, Masson et Cie, Paris, France, 1961, Vol. I, p. 38.

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1"amm,J., Voigt, K. D., and Volkwein, U., STEROIDS,_2, 271 (1963).

1

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Beling, C. G~ACTA ENDOCRINOL., Suppl 79 (1963). Kushinsky, S., and Tang, J. W., ACTA ENDOCRINOL., 43, 345 (1963). Zumoff, B., and Bradlow, H. L., J. CIIN. ENDOCR., 23._ 2 799 (1963). Kornel, L., J. CLIN. ENDOCR., 23__,1192 (1963).

8.

Arcos, M., and Ueberman, S., J. CIIN. ENDOCR., 25__,808 (1965).

9.

Gupta, D., and Tanner, J. M., BtOCHEM. J., 96, 25 P (1965).

10.

Gupta, D., and Goodwin, J., STEROIDS, 8, 195 (1966).

11.

Fukushima, D. K., Gallagher, T. F., Greenberg, W., and Pearson, O. H., J. CIIN. ENDOCR., 20, 1234 (1960).

12.

Herberg, R. J., ANAL. CHEM., 32__,42 (1960).